U.S. patent number 5,993,633 [Application Number 08/904,419] was granted by the patent office on 1999-11-30 for capillary electrophoresis electrospray ionization mass spectrometry interface.
This patent grant is currently assigned to Battelle Memorial Institute. Invention is credited to Joanne C. Severs, Richard D. Smith.
United States Patent |
5,993,633 |
Smith , et al. |
November 30, 1999 |
**Please see images for:
( Certificate of Correction ) ** |
Capillary electrophoresis electrospray ionization mass spectrometry
interface
Abstract
The present invention is an interface between a capillary
electrophoresis separation capillary end and an electrospray
ionization mass spectrometry emitter capillary end, for
transporting an anolyte sample from a capillary electrophoresis
separation capillary to a electrospray ionization mass spectrometry
emitter capillary. The interface of the present invention has: (a)
a charge transfer fitting enclosing both of the capillary
electrophoresis capillary end and the electrospray ionization mass
spectrometry emitter capillary end; (b) a reservoir containing an
electrolyte surrounding the charge transfer fitting; and (c) an
electrode immersed into the electrolyte, the electrode closing a
capillary electrophoresis circuit and providing charge transfer
across the charge transfer fitting while avoiding substantial bulk
fluid transfer across the charge transfer fitting. Advantages of
the present invention have been demonstrated as effective in
providing high sensitivity and efficient analyses.
Inventors: |
Smith; Richard D. (Richland,
WA), Severs; Joanne C. (Hayward, CA) |
Assignee: |
Battelle Memorial Institute
(Richland, WA)
|
Family
ID: |
25419127 |
Appl.
No.: |
08/904,419 |
Filed: |
July 31, 1997 |
Current U.S.
Class: |
204/601; 204/450;
250/288; 977/882 |
Current CPC
Class: |
G01N
27/44717 (20130101); H01J 49/167 (20130101); Y10S
977/882 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 027/26 () |
Field of
Search: |
;204/450,603
;7/451,452,601 ;436/173,174 ;250/288 |
Other References
Severs et al. ("A New High-perfromance Interface for Capillary
Electrophoresis/Electrospray ionization Mass Spectrometry", Rapid
Communications in MAss Spectrometry, vol. 10, 1175-1178, month
unknown. 1996). .
Lamoree et al. ("On-line coupling of micellar electrokinetic
chromatography to electrospray mass spectrometry", Journal of
Chromatography A, 712 (month unknown 1995) 219-2250 month unknown.
.
Lamoree et al. ("Use of heptakis92,6-di-o-methyl)B-cyclodextrin in
on-line capillary zone electrophoresis-mass spectrometry for the
chiral separation of ripivacaine", Journal of Chromathography A,
742 (month unknown 1996) 235-242). .
Liquid Junction Coupling for Capillary Zone Electrophoresis/Ion
Spray Mass Spectrometry, ED Lee, W Much, JD Henion, TR Covey,
Biomedical and Environmental Mass Spectromety, vol. 18, 844-850
(1989) Month unknown..
|
Primary Examiner: Warden; Robert
Assistant Examiner: Noguerda; Alex
Attorney, Agent or Firm: Zimmerman; Paul W.
Government Interests
This invention was made with Government support under Contract
DE-AC06 76RLO 1830 awarded by the U.S. Department of Energy. The
Government has certain rights in the invention.
Claims
We claim:
1. An interface between a capillary electrophoresis separation
capillary end and an electrospray ionization mass spectrometry
emitter capillary end with a gap therebetween, for transporting an
anolyte sample from a capillary electrophoresis separation
capillary to a electrospray ionization mass spectrometry emitter
capillary, the interface having:
(a) a reservoir containing an electrolyte surrounding both of said
capillary electrophoresis capillary end and said electrospray
ionization mass spectrometry emitter capillary end; and
(b) an electrode immersed into said electrolyte, said electrode
closing a capillary electrophoresis circuit and providing charge
transfer whereby a voltage is supplied for electrospraying; wherein
the improvement comprises:
a charge transfer fitting having a first end enclosing said charge
capillary electrophoresis capillary end, said charge transfer
fitting extending therefrom across said gap and having a second end
enclosing said electrospray ionization mass spectrometry emitter
capillary end, said charge transfer fitting permitting said charge
transfer from said electrode through said electrolyte across said
charge transfer fitting into said anolyte sample and avoiding
substantial bulk fluid transfer across said charge transfer
fitting.
2. The interface as recited in claim 1, wherein said electrolyte is
substantially electrochemically similar to said anolyte sample
thereby avoiding substantial ion transfer or bulk fluid transfer
across the charge transfer fitting.
3. The interface as recited in claim 1, wherein said charge
transfer fitting is a microdialysis tube.
4. The interface as recited in claim 1, wherein said electrospray
ionization mass spectrometry emitter capillary is less than or
equal to about 2 cm in length.
5. A method of providing an interface between a capillary
electrophoresis separation capillary end and an electrospray
ionization mass spectrometry emitter capillary end with a gap
therebetween, for transporting an anolyte sample from a capillary
electrophoresis separation capillary to a electrospray ionization
mass spectrometry emitter capillary, the method having the steps
of:
(a) surrounding both of said capillary electrophoresis capillary
end and said electrospray ionization mass spectrometry emitter
capillary end with an electrolyte; and
(b) immersing an electrode into said electrolyte, and closing a
capillary electrophoresis circuit thereby providing charge transfer
whereby a voltage is supplied for electrospraying; wherein the
improvement comprises:
placing a charge transfer fitting having a first end enclosing said
charge capillary electrophoresis capillary end, said charge
transfer fitting extending therefrom across said gap and having a
second end enclosing said electrospray ionization mass spectrometry
emitter capillary end, said charge transfer fitting permitting said
charge transfer from said electrode through said electrolyte across
said charge transfer fitting into said anolyte sample and avoiding
substantial bulk fluid transfer across said charge transfer
fitting.
6. The method as recited in claim 5, wherein said electrolyte is
substantially electrochemically similar to said anolyte sample
thereby avoiding substantial ion transfer or bulk fluid transfer
across the charge transfer fitting.
7. The method as recited in claim 5, wherein said charge transfer
fitting is a microdialysis tube.
8. The method as recited in claim 5, wherein said electrospray
ionization mass spectrometry emitter capillary is about 2 cm in
length.
9. A junction interface between a capillary electrophoresis
separation capillary end and an electrospray ionization mass
spectrometry emitter capillary end with a gap therebetween, for
transporting an anolyte sample from a capillary electrophoresis
separation capillary to a electrospray ionization mass spectrometry
emitter capillary, the interface having:
(a) a reservoir containing an electrolyte surrounding both the
capillary electrophoresis separation capillary end and the
electrospray ionization mass spectrometry emitter capillary end;
and
(b) an electrode immersed into said electrolyte, said electrode
closing a capillary electrophoresis circuit and providing charge
transfer from said electrolyte through said gap into said anolyte
sample, whereby a voltage is supplied for electrospraying;
wherein the improvement comprises:
a microdialysis tube having a first end enclosing the capillary
electrophoresis separation capillary end, extending across said gap
and enclosing the electrospray ionization mass spectrometry emitter
capillary end.
Description
FIELD OF THE INVENTION
The present invention relates generally to an interface between a
capillary electrophoresis (CE) separation capillary and an
electrospray ionization (ESI) mass spectrometry (MS) emitter
capillary.
BACKGROUND OF THE INVENTION
Interfacing between a CE to an ESI-MS is increasingly used in
biomedical and biochemical applications with minute sample volumes
and high-speed analyses. Several approaches for interfacing (i.e.
establishing electrical contact at or near the end of the CE
capillary) have been done.
In U.S. pat. No. Re. 34,757, 1994 (4,885,076, 1989), shows a CZE
with a high voltage power supply in series with an EIS high voltage
power supply spraying into an MS skimmer. Although high sensitivity
and efficiency was achieved, disadvantages included (1) a
dependence on the buffer system used, (2) ESI instability under
some operating conditions and (3) the need to regularly replace the
metal coating on the capillary tip.
In U.S. pat. No. 5,423,964, 1995 to Olivares et al., also spraying
into a skimmer, a metal coating on the tip of the CE capillary made
contact with a metal sheath capillary to which the CE terminus/ESI
voltage was applied. In addition, a non-conductive capillary in
combination with applying the EIS high voltage to a co-axial sheath
flow was used. The sheath liquid, with a small electrolyte content,
is infused to the ESI source through a sheath capillary surrounding
the end of the separation capillary and terminating near the end of
the separation capillary. The sheath liquid flowing at a few
microliters per minute is added to the CE effluent as it elutes
from the terminus of the CE capillary thereby providing the
necessary electrical contact. The sheath-liquid interface has been
widely employed, but not without shortcomings. The sheath-liquid
composition must be carefully selected to avoid formation of moving
ionic boundaries inside the capillary from migration of
sheath-liquid counterions into the CE capillary. These ionic
boundaries may cause variation in migration time and resolution. In
addition, the sheath liquid incorporates impurities and other
charge carrying species that can be ionized by the electrospray
process and decrease overall sensitivity.
A sheathless design has been reported by SA Hofstadler, F D Swanek,
J C Severs, A G Ewing and R D Smith, Rapid Commun. Mass Spectrom.,
"Analysis of Single Cells with Capillary Electrophoresis
Electrospray Ionization Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry", S. A. Hofstadler, J. C. Severs, R. D. Smith, F.
D. Swanek and A. G. Ewing, Rapid Commun. Mass Spectrom., 10,
919-922 (1996), wherein the capillary terminus was tapered and
coated with gold. Fabrication of the gold-coated, tapered
capillaries adds time and complexity to obtaining MS results. A
flow-rate minimum limit of approximately 70 nL/min has been
reported for the sheathless interface with improvements obtained if
run at higher flow-rates (up to 250 nL/min), Bateman, K.; Thibault,
P.; White, R. presented at the 44th ASMS Conference of the American
Society for Mass Spectrometry and Allied Topics, Portland, Oreg.,
1996.
The paper by E. D. Lee, W. Muck, J. D. Henion and T. R. Covey,
Biomed. and Env. Mass Spectrom. 18, 844 (1989), discusses a liquid
junction as an interface. Electrical contact is established through
a liquid reservoir surrounding the junction of the separation
capillary and a "transfer" capillary. A gap between the two
capillaries is adjusted to about 10-20 micrometer permitting
sufficient make-up liquid from the reservoir to be drawn into the
transfer capillary and avoiding anolyte loss. Of course,
elecrophoretic separation is terminated at the discontinuity of the
gap. The flow of make-up liquid into the "transfer" capillary is
induced by a pressure difference (generally arising due to some
combination of differences in height of the two ends of the
capillary and the venturi effect of the nebulizing gas at the
capillary tip). One of the disadvantages of the liquid junction
interface is the difficulty in establishing a reproducible spacing
of the capillary segments, and the fact that both the anolyte
transfer efficiency and the flow rate of added liquid from the
reservoir are critically dependent on this spacing.
A disadvantage of both the sheath-flow and liquid junction
interfaces is the need for electrolyte added to the ESI source
(emitter) to maintain the electrical circuit. This added
electrolyte often decreases biological or chemical molecule
detection sensitivity. And, as stated previously, disadvantages of
the sheathless gold-coated, tapered capillary include the
complexity of assembly, and a special terminus on the CE capillary
is required. Accordingly, there is a need for CE-ESI-MS interface
that is simple to assemble and operate, specifically neither
requiring gold plating nor requiring added electrolyte (buffer),
but permitting operating at low flow rates for a wide range of
liquid compositions.
SUMMARY OF THE INVENTION
The present invention is an interface between a capillary
electrophoresis separation capillary end and an electrospray
ionization mass spectrometry emitter capillary end, for
transporting an anolyte sample from a capillary electrophoresis
separation capillary to a electrospray ionization mass spectrometry
emitter capillary. The interface of the present invention has:
(a) a charge transfer fitting enclosing both of the capillary
electrophoresis capillary end and the electrospray ionization mass
spectrometry emitter capillary end;
(b) a reservoir containing an electrolyte surrounding the charge
transfer fitting; and
(c) an electrode immersed into the electrolyte, the electrode
closing a capillary electrophoresis circuit and providing charge
transfer across the charge transfer fitting while avoiding
substantial bulk fluid transfer across the charge transfer
fitting.
As used herein, the term "charge transfer fitting" is used to
describe an element that mechanically holds two capillary ends
together permitting flow from one capillary end into the other
longitudinally through the charge transfer fitting while at the
same time permitting electrical charge to pass transversely through
the fitting while avoiding bulk flow transversely through the
charge transfer fitting.
Advantages of the present invention have been demonstrated as
effective in providing high sensitivity and efficient analyses. The
present invention interface may operate with sharply tapered
emitters and with or without the addition of any `make-up` liquids,
thereby increasing sensitivity and eliminating ionic boundary
formation under normal operation. The ability to operate at lower
flowrates relative to the `sheathless` interface has also been
demonstrated, providing the capability for operation on-line with a
wider range of CE conditions for separation optimization.
The present invention also avoids the need to coat the
electrophoretic capillary terminus with a metal contact, greatly
reducing capillary preparation time and increasing flexibility in
the use of different capillaries. The placement (e.g., aligning and
gluing) of the two capillaries inside the micro-dialysis tube is
simple (easily or readily accomplished), reproducible and
inexpensive. Additionally, the components of separation capillaries
and electrospray emitters may now be optimized independently, with
the small etched emitters readily produced in batches. The tips,
detached from the CE voltage via a junction, also last longer than
their conductive gold-tipped counterparts, and are less prone to
problems arising from electrical breakdown. Acidification of the CE
effluent has also been demonstrated for providing post-separation
solution changes.
As well as providing improved sensitivity in the higher m/z range
relative to the `sheathless` interface, the interface of the
present invention maintains effective ESI interface operation at
lower CE flow-rates, and therefore operates with a broader range of
CE separation conditions.
It is an object of the present invention to provide a reliable,
flexible, and rugged CE-ESI-MS interface.
The subject matter of the present invention is particularly pointed
out and distinctly claimed in the concluding portion of this
specification. However, both the organization and method of
operation, together with further advantages and objects thereof,
may best be understood by reference to the following description
taken in connection with accompanying drawings wherein like
reference characters refer to like elements.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a cross section of the interface of the present
invention.
FIG. 2 is a set of mass electropherograms for a CE-ESI-MS
separation of a set of benzenesulphonamides (MWs.: 329, 359, 385,
400, 419), employing a micro-dialysis membrane tubing junction.
Background electrolyte=20 mM NH.sub.4 OAc (pH 8.3), 30 kV,
selected-ion-monitoring.
FIG. 3a is a reconstructed ion electropherogram from a CE-ESI-MS
full scan analysis (m/z 1700-3300) of BCA II.
FIG. 3b is a mass spectrum of BCA II when junction
electrolyte=background electrolyte=20 mM NH.sub.4 OAc (pH 8.3).
FIG. 3c is a mass spectrum of BCA II when junction electrolyte=3%
acetic acid.
FIG. 4a is a protein mixture mass electropherogram obtained with a
micro-dialysis junction (charge transfer fitting) with separation
conditions of 30 micrometer ID.times.1.1 m APS coated capillary, 10
mM HOAc, 30 kV, m/z 1000-2600 in 2 sec.
FIG. 4b is a protein mixture mass electropherogram obtained with a
coaxial sheath-flow junction (75:24:1 IPA:H.sub.2 O:HOAc at 1
microliter per minute) with separation conditions of 30 micrometer
ID.times.1.1 m APS coated capillary, 10 mM HOAc, 30 kV, m/z
1000-2600 in 2 sec.
FIG. 5a is a mass electropherogram of benzenesulphonamides with a
UV detection at 200 nm.
FIG. 5b is a mass electropherogram of benzenesulphonamides with
ESI-MS detection over m/z 320-430 with a charge transfer
fitting.
FIG. 6a is a mass spectrum of a charge ladder of bovine carbonic
anhydrase II sample 15 mg/mL total protein with separation
conditions of 30 kV for 3 min, reduced to 15 kV for remainder of
run, 10 mM NH.sub.4 OAc (pH 9), scanning m/z 2900-3390 at 1
sec/scan.
FIG. 6b is a mass electropherogram of a charge ladder of bovine
carbonic anhydrase II sample 15 mg/mL total protein with separation
conditions of 30 kV for 3 min, reduced to 15 kV for remainder of
run, 10 mM NH.sub.4 OAc (pH 9), scanning m/z 2900-3390 at 1
sec/scan.
FIG. 7a is a mass electropherogram of a separation of
benzenesulphonamides with a first interface made according to the
present invention.
FIG. 7b is a mass electropherogram of a separation of
benzenesulphonamides with a second interface made according to the
present invention.
FIG. 7c is a mass electropherogram of a separation of
benzenesulphonamides with a third interface made according to the
present invention.
FIG. 8a is a mass spectrum of FMAF with separation conditions using
polyacrylamide coated capillary and 10 mM NH.sub.4 OAc (pH 7.9), 30
kV.
FIG. 8b is a mass electropherogram of FMAF with separation
conditions using polyacrylamide coated capillary and 10 mM NH.sub.4
OAc (pH 7.9), 30 kV.
FIG. 9a is a mass electropherogram of horse-skeletal muscle
myoglobin with a separation electrolyte=junction electrolyte=10 mM,
pH 7.9, NH.sub.4 OAc.
FIG. 9b is a mass spectrum of horse-skeletal muscle myoglobin with
a separation electrolyte=junction electrolyte=10 mM, pH 7.9,
NH.sub.4 OAc.
FIG. 10a is a mass electropherogram of horse-skeletal muscle
myoglobin with a separation electrolyte=10 mM, pH 7.9, NH.sub.4
OAc, and a junction electrolyte=1% HOAc.
FIG. 10b is a mass spectrum of horse-skeletal muscle myoglobin with
a separation electrolyte=10 mM, pH 7.9, NH.sub.4 OAc, and a
junction electrolyte=1% HOAc.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
Referring to FIG. 1, the present invention is an interface 100
between a capillary electrophoresis separation capillary end 102
and an electrospray ionization mass spectrometry emitter capillary
end 104, for transporting an anolyte sample from a capillary
electrophoresis separation capillary 106 to a electrospray
ionization mass spectrometry emitter capillary 108. The anolyte
sample is made up of a background electrolyte and a biological or
chemical molecule. Background electrolyte may be any electrolyte
including but not limited to acetic acid, ammonium acetate,
ammonium carbonate, and combinations thereof. Biological molecules
amenable to analysis include but are not limited to carbohydrates,
glycoproteins, peptides, and proteins, nucleotides,
ologonucleotides, and DNA-protein complexes, DNA segment, synthetic
polymers biopolymers and combinations thereof.
The interface 100 has a charge transfer fitting 110 enclosing both
of the capillary electrophoresis capillary end 102 and the
electrospray ionization mass spectrometry emitter capillary end
104. The interface 100 further includes a reservoir (not shown)
containing an electrolyte 112 surrounding the charge transfer
fitting 110, and an electrode 114 immersed into the electrolyte
112, the electrode 114 closing a capillary electrophoresis circuit
(not shown) and providing charge transfer transversely across the
charge transfer fitting from the electrode 114 through the
electrolyte 112 and through the charge transfer fitting 110 to the
anolyte sample 116. The charge transfer fitting 110 may permit
transverse charge transfer in part or in whole over its surface
area. The electrolyte 112 may be any electrolyte having a
conductivity appropriate for CE including but not limited to acetic
acid, ammonium acetate, ammonium carbonate, and combinations
thereof. Preferred are more volatile electrolytes, e.g. ammonium
acetate to obtain enhanced performance.
The electrospray ionization mass spectrometry emitter capillary 108
is preferably less than about 10 cm in length and more preferably
less than 3 cm in length. Emitters 108 with lengths greater than 3
cm were found to be less reliable in providing a constant flow to
the ESI source. Shortening the length to 2 cm improved
performance.
The charge transfer fitting 110 may be any fitting permitting
charge transfer from the electrolyte 112 to the anolyte sample 116.
In a preferred embodiment, the charge transfer fitting 110 is
microdialysis tubing. It is further preferred that the electrolyte
112 is substantially electrochemically similar to the anolyte
sample 116 in order to avoid substantial bulk fluid transfer across
the charge transfer fitting 110. Electrochemically similar means
that the anolyte sample 116 and the electrolyte 112 do not
substantially transfer ions or bulk fluid under an electrical
potential. Substantial ion transfer or bulk fluid transfer is
sufficient to affect MS analysis results.
The reservoir may be closed or open, or of any material, but an
open reservoir is preferred, rather than an enclosed/limited
reservoir, and plastic is preferred rather than metal, to avoid the
production of air bubbles in the electrolyte circuit.
The high voltage power supply (HVPS) (not shown) to the ESI
interface is connected to the electrode 114, preferably copper
wire. A common ground connection is also formed between the CE
system, the outer casing of the ESI HVPS lead and the mass
spectrometer. Because the mass spectrometer does not act as a
current sink, previous attempts at constructing CE-ESI-MS
interfaces have occasionally resulted in damaging discharges to the
ESI system when the high voltage was applied and the gold contact
was disturbed. In the present invention, the potential across the
CE circuit terminates in a stable electrical contact prior to the
ESI emitter, thereby avoiding the damaging discharges.
Another important feature of this interface 100 is that it does not
distort or alter the CE separation. Under routine operating
conditions the electrolyte outside the dialysis membrane (i.e., in
the pipette tip) was the same as the CE background electrolyte in
the anolyte sample, so there was no net ion transfer across the
membrane, and no resulting ionic boundaries propagated into the
separation capillary. However, the effect of purposefully changing
the electrolyte in the reservoir, to change the solution conditions
post-run, can also be exploited based upon this interface. The
ability to acidify anolytes after a separation under neutral
conditions, for example for the study of non-covalent interactions
or capillary isoelectric focusing, is an attractive feature.
Example 1
An experiment was conducted to demonstrate the present
invention.
Reagents and Materials. Deionized distilled water from a Nanopure
II water system (Barnstead, Dubuque, Iowa) was used to prepare the
electrolyte 112 surrounding the charge transfer fitting 110 and
anolyte sample 116. Ammonium acetate was prepared from ammonium
hydroxide and glacial acetic acid (Sigma, St. Louis, Mo.), and
adjusted to the desired pH.
Fused silica capillaries of dimensions 192 .mu.m o.d.times.30 .mu.m
i.d..times.70 cm, obtained from Polymicro Technologies Inc.
(Phoenix, Ariz.), were employed for the capillary electrophoresis
separation capillary 106 and the electrospray ionization mass
spectrometry emitter capillary 108. The polyimide coating was
removed from the last 2 cm of short lengths of silica capillary,
and these portions were then etched in 40% hydrofluoric acid
(Aldrich, Milwaukee, Wis.) for approximately 30 minutes. The
resulting capillary was trimmed to produce a sharp emitter.
The charge transfer fitting 110 was a 250 .mu.m i.d. polysulphone
dialysis tubing (nominal molecular-weight cutoff of 10,000)
obtained from A/G Technology Corporation (Needham, Mass.).
The capillary electrophoresis separation capillary 106 and the 2 cm
long ESI mass spectrometer emitter capillary 108 were butted
together inside a 1.5 cm length of the charge transfer fitting 110
and epoxy (not shown) was then applied around the outside of the
charge transfer fitting/capillary boundaries forming the interface
100. After the epoxy had dried the interface 100 was inserted
through a 250 .mu.L eppendorf pipette tip (reservoir) containing
the electrolyte 112. These plastic tips successfully hold liquid
(electrolyte) even when positioned horizontally. The pipette tip
was connected to an x-y-z rotating insulating stand, and a copper
wire (electrode 114), attached to the stand, was extended into the
electrolyte 112.
Instrumentation. A, Crystal 310 CE system (ATI Unicam, Madison,
Wis.) was interfaced to a Finnigan TSQ 7000 triple quadrupole mass
spectrometer equipped with an electrospray ionization interface
(Finnigan MAT, San Jose, Calif.), employing a micro-spray
ionization source as shown and described in D. C. Gale and R. D.
Smith, Rapid Commun. Mass Spectrom., 7, 1017, hereby incorporated
by reference.
The spectrometer was tuned and calibrated using an acidic solution
of myoglobin and FMAF infused at 0.3 .mu.l/min. to the micro-spray
source. The electron multiplier was set to 1.3 kV and the
conversion dynode to -15 kV. The heated desolvation capillary in
the ESI source was held at 150.degree. C. CE-MS spectra were
acquired either full-scan (2 seconds per scan) or using
selected-ion-monitoring with a total step-cycle time of 1
second.
The high voltage power supply (HVPS) to the ESI interface was
connected to the copper wire and a common ground connection was
also formed between the CE system, the outer casing of the ESI HVPS
lead and the TSQ 7000 system.
An amount of the electrolyte 112 was pressurized through the
separation capillary for a few minutes to condition the walls and
the ESI voltage was applied and optimized at 1.6 kV before
injecting the anolyte sample and applying the separation
voltage.
Anolyte sample was bovine carbonic anhydrase II purchased from
Sigma, and the small benzenesulphonamide library was provided by
Prof. G. Whitesides, Harvard, (C. Liu, Q. Wu, A. C. Harms and R. D.
Smith, Anal Chem., (submitted)).
When the separation voltage was applied with the present design a
completed circuit was immediately observed, as characterized by a
rise in the ESI current observed on the TSQ monitor. At no time
were disruptions in the CE circuit ever observed; i.e., the
electrical connection across the membrane was reliably maintained.
Occasionally the ESI process faltered due to a blockage of the tip.
This was effectively remedied by filtering the buffers and
electrolyte solutions and could usually be reversed by placing the
tip in water for a few minutes. Unlike the liquid-junction
interface, there was no need to optimize a gap between the two
capillaries; the capillaries were simply pushed together as no
inlet flow of liquid or electrolyte through the junction was
necessary.
The separation and detection of some small benzenesulphonamides
with different amino acid tails, illustrated in FIG. 2,
demonstrates clearly that this new interface can provide high
efficiency separations. The CE peaks correspond to up to 350,000
theoretical plates. This analysis, conducted using a pH 8.5
ammonium acetate solution, demonstrates the generally obtainable
low femtomole to high attomole level sensitivity, (a sample volume
of 25 nL having 0.25 .mu.g/mL concentrations initially being
injected). Under acidic conditions even higher sensitivities were
achieved. It is important to note that at no time, even under the
low electroosmotic flow conditions using a pH 4.5 ammonium acetate
solution, was the use of a pressure gradient across the separation
capillary or across the membrane necessary to maintain the electric
circuit and a stable electrospray. Only species from the CE
separation were observed to be introduced to the electrospray
source.
The electrospray process was also maintained even prior to the
application of the separation voltage when an extremely sharp tip
was employed. Because the etched emitters did not have to be
exposed to refluxing and coating procedures, some extremely
fine/sharp capillary tips were be employed. Even under infusion
conditions it was seen that these finer emitters provided much more
stable electrospray signals extending much higher in the m/z range,
than obtained in our previous experience with gold-coated emitters.
This improved sensitivity and also facilitated identification of
proteins.
Example 2
An experiment was conducted wherein the anolyte sample was
electrochemically dis-similar to the electrolyte. Chemicals and
instrumentation were as in Example 1.
Bovine carbonic anhydrase II (BCA II) was injected from an initial
concentration of 2 mg/mL in water, run with a background
electrolyte of 20 mM ammonium acetate (pH 8.5) as an anolyte
sample. Electrolyte varied from identical to the background
electrolyte, to 3% acetic acid.
A CE-MS analysis (FIG. 3b) shows the mass spectrum obtained from
under the major peak in FIG. 3a when the electrolyte in the
reservoir was the same as the background electrolyte. FIG. 3c shows
the resulting spectrum, in which a charge-state distribution shift
can be observed, when the reservoir was changed to 3% acetic acid.
Use of the 3% acetic acid resulted in a three minute delay in
migration time, demonstrating an ionic boundary effect. This
experiment clearly demonstrates the potential for manipulation of
the external reservoir composition to manipulate both the
separation and the ESI process.
Example 3
Additional experiments were conducted to demonstrate CE-ESI-MS for
various anolyte samples in comparison to standard sheath flow
methods. The reagents, materials and instrumentation were as in
Example 1.
The separation and emitter capillaries were precoated internally
with aminopropyltrimethoxysilane (Aldrich, Milwaukee, Wis.) to
avoid anolyte interaction with the charged capillary walls, when
separating protein mixtures. In cases where elimination of the
electroosmotic flow (EOF) through the capillaries was also desired,
the capillaries were precoated externally with
methacryloxypropyltrimethoxysilane (Aldrich) and then
polyacrylamide (BRL Life Technologies, Gaithersburg, Md.).
Standard proteins and peptides were purchased from Sigma, and the
small benzenesulphonamide library (Gao, J.; Cheng, X.; Chen, R.;
Sigal, G. B.; Bruce, J. E.; Schwartz, B. L.; Hofstadler, S. A.;
Anderson, G. A.; Smith, R. D.; Whitesides, G. M. J. Med. Chem.,
1996, 39, 1949-1955) and bovine carbonic anhydrase II (BCA II)
charge ladder (Gao, J.; Gomez, F. A.; Haerter, R.; Whitesides, G.
M. Proc. Natl. Acad. Sci. USA 1994, 91, 12027-12030) were provided
by the laboratory of Prof. G. Whitesides, Harvard.
The micro-spray ionization source of Example 1 was used when using
the interface 100 of the present invention and the standard
Finnigan source when using a coaxial sheath flow system. The sheath
flow, composed of 75:24:1 isopropanol:water:acetic acid, was
infused via a Harvard syringe pump at a flow rate of 1 .mu.L/min.
The spectrometer was tuned and calibrated using an acidic solution
of myoglobin and FMAF infused at 0.3 .mu.l/min. through the
micro-spray source. The electron multiplier was set to 1.3 kV and
the conversion dynode to -15 kV. The heated desolvation capillary
in the ESI source was held at 160.degree. C. CE-MS spectra were
acquired either full-scan (1 or 2 seconds per scan) or using
selected-ion-monitoring with a total step-cycle time of 1
second.
For these tests, the flow-rate at the ESI emitter with the
micro-dialysis junction interface was nearly the same as the
flow-rate through the CE capillary (which is generally in the range
of nL/min), demonstrating or confirming no significant mass
transfer through the dialysis membrane, only ionic transfer.
Moreover, the electrolyte in the reservoir was the same as the
background electrolyte in the anolyte sample resulting in no net
electrolyte addition, no anolyte ion dilution and no production of
ionic boundaries.
Three parameters of primary importance for the performance of a
CE-MS interface were analyzed. These are: (1) sensitivity, (2)
separation efficiency, and (3) reliability or ruggedness of the
interface.
Sensitivity: FIGS. 4a and 4b demonstrates the surprising gain in
sensitivity achieved employing the interface 100 (micro-dialysis
junction) (FIG. 4a) for separation of a 10 .mu.M protein mixture
relative to that achieved employing the sheath-flow interface (FIG.
4b). Care was taken to ensure that equivalent separation parameters
were employed in both cases, capillaries were conditioned
identically and the same sample volumes were injected. For this
comparison, in which the analyzer was scanning m/z 1000-2600 at 2
sec/scan, the sheath-flow was optimized and minimized to 1
.mu.L/min (2-5 .mu.L/min sheath-flows are generally reported). An
optimized spray voltage of 3.7 kV was applied for ionization. The
detection limit for the proteins under these full-scan conditions
was approximately 10 .mu.M when employing the sheath-flow system,
but the biological or chemical molecules could still be detected
when diluted 10-fold employing the micro-dialysis junction. Our
studies to date indicate that approximately an order of magnitude
improvement in detection limits was achieved.
Separation Efficiency: FIGS. 5a and 5b shows a comparison of CE
with on-capillary UV detection at 200 nm (FIG. 5a) and CE-ESI-MS
employing the micro-dialysis interface (FIG. 5b) for a separation
of several benzenesulphonamides. As can be seen, there is only a
small loss in separation efficiency (<20%) and comparable
sensitivity (full scan) is obtained. The specificity of mass
spectral detection, however, means that any small loss of
separation efficiency is generally unimportant. Although there is a
slight efficiency loss relative to on-line UV detection, it should
also be noted that efficiency retention is generally comparable to
other mass spectral interfaces, as demonstrated in FIG. 4b for the
sheath-flow interface.
FIGS. 6a and 6b shows a separation of a charge ladder of bovine
carbonic anhydrase II. Each peak in this separation is due to an
extra conversion of a positively-charged Lys
.epsilon.--NH.sub.3.sup.+ group on the protein surface to a neutral
N-acyl derivative; the mixture is a set of protein derivatives
differing in charge in solution by integral units. The CE
separation due to the charge on the protein derivatives was
maintained through the interface. Additionally, the ability to
spray from a purely aqueous solution, without post-run addition of
a sheath-flow, allowed the mass spectral analysis of native
proteins at neutral pH conditions. The gold-coated `sheathless`
interface would also be expected to provide comparable performance,
however, we found that some gold-coated tips, which are more
difficult to make as sharply tapered as the tips now being employed
(due to their need to withstand refluxing and coating procedures),
did not provide useful mass spectra in the higher m/z range. In
contrast, this experiment demonstrates that useful data can be
readily obtained across the m/z range 1000-4000 using the
micro-dialysis junction of the present invention under aqueous
conditions.
Reliability: A stable electrical circuit has been immediately
achieved with each junction so far constructed. For emitter
capillaries limited to lengths of <2 cm, the reliability of this
interface is significantly improved compared to other interfaces.
As there is no potential difference across the emitter capillary,
liquid flow through this short capillary section depends upon the
EOF generated in the separation capillary, the pressure applied by
the separation capillary eluant and any flow induced by the
electrospray process. These parameters appear to not provide a
sufficient flow through an emitter of length greater than 2 cm.
Blockage of the 30 .mu.m i.d. emitter tips is rare and can be
further minimized by filtering electrolytes and storing tips in
distilled water. (The tips can often be restored by careful
trimming with a capillary cutter, if necessary.) Smaller diameter
tips were investigated but were found to block too readily for
routine utility.
A difference in migration times is always noted between UV,
sheath-flow ESI-MS and micro-dialysis junction ESI-MS detection due
to the differences in electric fields and boundary effects.
However, a relatively large variation in migration times through
different CE capillaries is also common if they are not fully
conditioned. FIGS. 7a, 7b and 7c illustrate the reproducibility
obtained from three separately constructed interfaces (i.e. three
different capillaries and tips). The three interfaces were
essentially identical according to the present invention. For this,
no time was expended in optimizing relative capillary positions and
flow-rates, but relatively good reproducibility (within CE-MS
standards) was still achieved. However, the third separation has a
lower separation efficiency, an observation attributed to poor
capillary cuts at the junction.
Table 1 shows average percentage coefficients of variation in
migration time, peak area and peak efficiency for the first,
fourth, and fifth peaks for 3 runs obtained using a single
capillary and for 3 runs obtained with 3 different capillaries.
These results were achieved despite little time being spent in
capillary conditioning; also results were calculated from only a
small number of runs and no internal standard was applied.
TABLE 1 ______________________________________ Comparison of
coefficients of variation (c. of v.) in migration times, efficiency
and peak areas calculated from 3 runs using (A) a single capillary
and 3 runs using (B) 3 different capillaries. Migration Time
Efficiency Peak Area (% c. of v.) (% c. of v.) (% c. of v.)
______________________________________ A 3.1 13 20 B 3.6 38 37
______________________________________
FIGS. 8a and 8b show the mass spectrum (FIG. 8a) and mass
electropherogram (FIG. 8b) of the peptide FMAF (MW=598) obtained
using a polyacrylamide coated capillary. The polyacrylamide
neutralizes the negative charge on the silica capillary walls and
therefore minimizes the EOF. CE-MS under these very low flow-rate
conditions employing a sheathless interface would not have been
feasible. The stability of the polyacrylamide coating is known to
be limited, especially under extremes of pH. However, the
polyacrylamide was active during this particular run as over the
coarse of the following few analyses, evidenced by a steady rise in
the electrospray current and the background signal was observed as
the coating degenerated and EOF increased.
Example 4
An experiment was conducted to demonstrate a further advantage of
the interface 100 (micro-dialysis junction), specifically
post-column solution changes, or the ease with which the
composition of the electrolyte in the junction reservoir can be
changed to manipulate the separation, the ESI process or the
anolyte. This can be useful for capillary isoelectric focusing and
capillary isotachophoresis as well as post-run acidification to aid
the ESI process or to study non-covalent interactions. We have
previously demonstrated that it is possible to shift the
charge-state distribution of a protein by this technique, however,
it was not clear that solution conditions could be altered
sufficiently to disrupt a noncovalent complex in this on-line
manner.
FIGS. 9a and 9b show the mass electropherogram (FIG. 9a) and mass
spectrum (FIG. 9b) obtained for horse-skeletal-muscle myoglobin
when the electrolyte in the CE capillary and in the reservoir was
composed of 10 mM ammonium acetate (pH 7.9). The anolyte peak is
relatively broad under these non-optimized separation conditions.
The intact holomyoglobin complex was maintained throughout the
separation and `soft` ESI process.
FIGS. 10a and 10b show the mass electropherogram (FIG. 10a) and
mass spectrum (FIG. 10b) obtained when the electrolyte in the
reservoir was changed to 1% acetic acid. In this case the myoglobin
migrated through the capillary as the intact complex, but on
traversing the junction was denatured, is denatured and loses the
heme moiety. The mass spectrum using the acidic junction conditions
is clearly more intense and shows a predominance of ions due to the
denatured protein.
CLOSURE
While a preferred embodiment of the present invention has been
shown and described, it will be apparent to those skilled in the
art that many changes and modifications may be made without
departing from the invention in its broader aspects. The appended
claims are therefore intended to cover all such changes and
modifications as fall within the true spirit and scope of the
invention.
* * * * *